Flow cytometer systems are used to detect and count microorganisms and in varied applications throughout the life sciences including clinical diagnostics and immunology, protein and nucleic acid detection, hematology, and oncology. Commercially-available instruments range from complex laboratory systems that may be configured for a wide range of measurements to low-cost bench top systems with more limited capabilities. In the current biotechnology market, the price of a flow cytometer typically increases with its measurement precision and the number of different measurements it is capable of performing.
Flow cytometers are typically used to identify and count particles with specific characteristics in a fluid sample. As used herein, the term “particles” refers to, for example, latex spheres, bacteria, viruses, DNA fragments, cells, molecules, or constituents of whole blood. Particles may scatter excitation light directly or fluoresce when illuminated by light of an appropriate wavelength. In many cases, the fluorescent emission properties are optimized for specific measurements by attaching probe molecules to the entire particles or to microscopic structures within the particles.
In a typical flow cytometer, a particle-containing sample fluid flows through an excitation volume where the particles are illuminated by a focused light source. As they flow through the excitation volume, the particles scatter light out of the beam and fluoresce. In many cases, the fluorescent emission process is enhanced by bonding probe molecules to the particles or to structures within the particles. Particles are typically identified and counted by collecting and analyzing the light pulses that are emitted and scattered as the particles pass through the excitation volume.
Flow cytometers may be divided into two broad categories according to the composition of the fluid in and around the excitation volume. In sheath flow instruments, the fluid in the region of the excitation volume has two components: the particle-containing sample fluid and a particle-free sheath fluid that surrounds the sample fluid and confines it to a region near the flow axis. In capillary flow cytometers, the particle-containing sample fluid fills the entire flow volume and there is no sheath fluid.
Techniques for analyzing pulses produced by flow cytometers have been known and described for example in U.S. Pat. Nos. 4,021,117 and 3,938,038, the disclosures of which are incorporated herein by reference in their entirety.